Gram negative bacteria are the causative agents for a number of human pathologies and there is a need for effective vaccines to be developed against many of these bacteria. In particular Bordetella pertussis, Borrelia burgdorferi, Brucella melitensis, Brucella ovis, Chlamydia psittaci, Chlamydia trachomatis, Esherichia coli, Haemophilus influenzae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa and Yersinia enterocolitica are Gram negative bacteria which cause pathologies which could be treated by vaccination.
Neisseria meningitidis is an important pathogen, particularly in children and young adults. Septicemia and meningitis are the most life-threatening forms of invasive meningococcal disease (IMD). This disease has become a worldwide health problem because of its high morbidity and mortality.
Thirteen N. meningitidis serogroups have been identified based on antigenic differences in the capsular polysaccharides, the most common being A, B and C which are responsible for 90% of disease worldwide. Serogroup B is the most common cause of meningococcal disease in Europe, USA and several countries in Latin America.
Vaccines based on the capsular polysaccharide of serogroups A, C, W and Y have been developed and have been shown to control outbreaks of meningococcal disease (Peltola et al 1985 Pediatrics 76; 91-96). However serogroup B is poorly immunogenic and induces only a transient antibody response of a predominantly IgM isotype (Ala'Aldeen D and Cartwright K 1996, J. Infect. 33; 153-157). There is therefore no broadly effective vaccine currently available against the serogroup B meningococcus which is responsible for the majority of disease in most temperate countries. This is particularly problematic since the incidence of serotype B disease is increasing in Europe, Australia and America, mostly in children under 5. The development of a vaccine against serogroup B meningococcus presents particular difficulties because the polysaccharide capsule is poorly immunogenic owing to its immunologic similarity to human neural cell adhesion molecule. Strategies for vaccine production have therefore concentrated on the surface exposed structures of the meningococcal outer membrane but have been hampered by the marked variation in these antigens among strains.
Further developments have led to the introduction of vaccines made up of outer membrane vesicles which will contain a number of proteins that make up the normal content of the bacterial membrane. One of these is the VA-MENGOC-BC® Cuban vaccine against N. meningitidis serogroups B and C (Rodriguez et al 1999 Mem Inst. Oswaldo Cruz, Rio de Janeiro 94; 433-440). This vaccine was designed to combat an invasive meningococcal disease outbreak in Cuba which had not been eliminated by a vaccination programme using a capsular polysaccharide AC vaccine. The prevailing serogroups were B and C and the VA-MENGOC-BC® vaccine was successful at controlling the outbreak with an estimated vaccine efficiency of 83% against serogroup B strains of N. meningitidis (Sierra et al 1990 In Neisseria, Walter Gruyter, Berlin, m. Atchman et al (eds) p 129-134, Sierra et al 1991, NIPH Ann 14; 195-210). This vaccine was effective against a specific outbreak, however the immune response elicited would not protect against other strains of N. meningitidis. 
Subsequent efficacy studies conducted in Latin America during epidemics caused by homologous and heterologous serogroup B meningococcal strains have shown some efficacy in older children and adults but its effectiveness was significantly lower in younger children who are at greatest risk of infection (Milagres et al 1994, Infect. Immun. 62; 4419-4424). It is questionable how effective such a vaccine would be in countries with multistrain endemic disease such as the UK. Studies of immunogenicity against heterologous strains have demonstrated only limited cross-reactive serum bactericidal activity, especially in infants (Tappero et al 1999, JAMA 281; 1520-1527).
A second outer membrane vesicle vaccine was developed in Norway using a serotype B isolate typical of those prevalent in Scandinavia (Fredriksen et al 1991, NIPH Ann, 14; 67-80). This vaccine was tested in clinical trials and found to have a protective efficacy after 29 months of 57% (Bjune et al 1991, Lancet, 338; 1093-1096).
However, the use of outer membrane vesicles in vaccines is associated with some problems. For instance, the OMV contain toxic lipopolysaccharides (LPS). The toxicity of outer membrane vesicles may be decreased by treatment with detergents to remove the majority of LPS in order to prevent toxic reactions in vaccinees. This procedure unfortunately also removes other potentially important vaccine components such as surface exposed lipoproteins.
The imp gene encodes the Imp/OstA protein which is an outer membrane protein of Gram negative bacteria. Imp/OstA has been most extensively studied in E. coli where it was first described as having a role in outer membrane permeability (Sampson et al 1989 Genetics 122, 491-501). Imp/OstA was subsequently found to determine organic solvent tolerance in E. coli (Aono et al 1994 Appl. Environ. Microbiol. 60, 4624-4626). It has been proposed that Imp/OstA contributes to n-hexane resistance of E. coli by reducing the influx of n-hexane (Abe et al 2003, Microbiology 149, 1265-1273).
The msbA gene was first identified in E. coli as a multicopy-suppressor of the mutation in the htrB (lpxL) gene, which encodes an enzyme involved in a late step of lipid A biosynthesis (Karow and Georgeopoulos, 1993. Mol. Microbiol. 7, 69-79). The MsbA protein belongs to a family of ABC (ATP-binding cassette) transporters. A temperature-sensitive msbA mutant of E. coli has been reported to accumulate LPS as well as three major PL in the inner membrane when shifted to the restrictive growth temperature (Doerrler, et al 2001 J. Biol. Chem. 276, 11461-11464). This result indicated a role for MsbA in the translocation of both LPS and PL across the inner membrane and/or, as proposed earlier (Polissi and Georgopoulos, 1996 Mol. Microbiol. 20, 1221-1233), in a later step of the transport process.
There is a need for improved vaccines for use in treatment and prevention of Gram negative bacterial infection, particularly Neisserial infection. It is particularly important to address the problem of LPS toxicity in vaccines comprising whole bacteria, or outer membrane vesicle preparations whilst ensuring that desirable antigens are retained in the outer membrane. The present application discloses the general concept of outer membrane vesicle vaccines prepared from Gram negative bacterial mutant strains, particularly Neisserial strains such as N. meningitidis, which have reduced LPS compared to wild type strains, or no LPS on its surface. Such vaccines have the advantage that the outer membrane vesicles may be produced using a protocol involving extraction with low or no detergent thus retaining protective antigens such as lipoproteins on the outer membrane vesicle surface. It is particularly preferred if a low level (less than 50, 40, 30, 20 or 10% of wild-type level) of LPS is maintained in the mutant strain so that one or both of the following advantages are realised: i) the LPS can still be used as an antigen in its own right, and ii) the strain may grow better for production purposes. The inventors have found that disruption of either the Imp or MsbA proteins can produce such strains and outer membrane vesicle vaccines. A particularly preferred mutant for these purposes is a functional disruption of the imp gene.
The present invention further provides a mutated Imp or MsbA protein, for example a chimeric protein comprising a backbone polypeptide which is derived from an Imp protein and at least one insert region derived from a different protein wherein part or all of at least one Imp extracellular loop is replaced with one or more polypeptide sequence from at least one additional protein. Also provided are vaccine components comprising a chimera of part or all of at least one Imp extracellular loop with a different carrier protein which provides T-helper epitopes.
The present application discloses proteins that regulate the transport of LPS to the outer membrane of Gram negative bacteria. In particular, a function has been provided for Imp in regulating the transport of LPS to the outer membrane of Gram negative bacteria. It further discloses that MsbA regulates the transport of LPS to the outer membrane of Neisseria and the disruption of this protein does not lead to a disruption of phospholipid transport to the outer membrane. Downregulation of Imp or MsbA, either by downregulation of expression of the imp or msbA gene or by disrupting the structure of the Imp or MsbA protein so that it no longer transports LPS to the outer membrane, leads to most (but not all) of the LPS failing to reach the cell surface as shown in FIGS. 5 and 10. Downregulation of Imp MsbA also leads to a decrease in the amount of LPS present in the bacteria due to feedback inhibition on LPS synthesis by mislocalised LPS. Downregulation of Imp or MsbA therefore produces a Gram negative bacterium (preferably a Neisserial bacterium) with a low level of LPS, equivalent or lower to the level achieved after detergent treatment. Such a bacterium has lower toxicity whilst retaining sufficient LPS to enable the LPS to contribute to the immunogenicity of the bacterium/vaccine composition.
A further advantageous aspect of some embodiments of the invention is that the Imp protein is used as a scaffold to display advantageous heterologous antigens on the outer membrane of Gram negative bacteria, preferably a Neisserial strain, more preferably N. meningitidis. These antigens are positioned at the site of one of the Imp extracellular (surface exposed) loops.
A further advantage of some embodiments of the invention is realised when at least some of the extracellular loops of Imp are retained in the chimeric protein of the invention. The amino acid sequence of the extracellular loops are well conserved and antibodies against an extracellular loop of Imp should crossreact with a wide range of bacterial, preferably Neisserial strains.
In a preferred embodiment, the invention provides a Gram negative bacterium in which a protein involved in the transport of LPS to the outer membrane, for instance Imp or MsbA, is down regulated such that LPS transport to the outer membrane is disrupted.
In a further embodiment, the invention provides a polynucleotide comprising a sequence encoding the mutated or chimeric protein of the invention, an expression vector comprising a sequence encoding the chimeric protein of the invention and a host cell comprising said expression vector. Polynucleotides of the invention do not encompass a bacterial genome.
In a further embodiment, the invention provides an outer membrane vesicle preparation, from a strain in which the expression of a protein regulating LPS transport to the outer membrane, for instance Imp or MsbA, is downregulated such that the outer membrane vesicle has a lower LPS content than outer membrane vesicles derived from a similar strain of Gram negative bacterium in which transport of LPS to the outer membrane has not been disrupted.
In a further embodiment, the invention provides a method for producing the chimeric protein or outer membrane vesicle preparation of the invention.
In a further embodiment, the invention provides a pharmaceutical preparation, preferably a vaccine comprising the Gram negative (preferably Neisserial) bacterium of the invention or a fraction or membrane thereof, the chimeric protein of the invention, or the outer membrane vesicle preparation of the invention, and a pharmaceutically acceptable carrier.
In a further embodiment, the invention provides methods of treatment or prevention of Gram negative bacterial infection, preferably Neisserial infection.